Cytomegalovirus pp65 - Recombinant Protein: A New Antigen for Efficient Restimulation of CD4⁺ and CD8⁺ pp65-Specific T Lymphocytes.

Short-term restimulation assays combined with the analysis of effector function, in particular the detection of cytokine production, are useful tools for the analysis and isolation of antigen-specific T cells. Until now, restimulations with soluble protein antigens failed to efficiently reactivate CD8⁺ T cells. We have developed a recombinant protein of the immunodominant cytomegalovirus (CMV) matrix protein pp65 for in vitro restimulation of pp65-specific CD4⁺ as well as CD8⁺ T cells. The efficiency of the CMV pp65 - Recombinant Protein to reactivate pp65-experienced CD4⁺ and CD8⁺ T cells and the specificity of the restimulated T cells were analysed.

PBMC from CMV seropositive donors were restimulated with CMV pp65 - Recombinant Protein or a complete pool of overlapping pp65 peptides. Afterwards T cells were analysed for intracellular IFN-γ production by flow cytometry. Interestingly, we observed that stimulation with CMV pp65 - Recombinant Protein results in IFN-γ production in CD4⁺ as well as CD8⁺ T cells with frequencies comparable to that using the peptide pool as antigen (n=17). In contrast, upon stimulation of PBMC from CMV seronegative donors with CMV pp65 - Recombinant Protein neither IFN-γ nor TNF-α were detectable in T cells (n=6).

Furthermore, we tested the specificity of CMV pp65 - Recombinant Protein-reactive CD4⁺ and CD8⁺ T cells. Therefore, IFN-γ-producing T cells were magnetically isolated after short-term stimulation with pp65 using the IFN-γ cytokine secretion assay and expanded for 7 days. Subsequently, the isolated and expanded CD4⁺ and CD8⁺ T cells were restimulated with pp65 peptide pool. More than 80 % of the CD4⁺ and CD8⁺ T cells produced IFN-γ and more than 80 % of the CD8⁺ T cells were positively stained with MHC class I/pp65 tetramers.

These results demonstrate that CMV pp65 - Recombinant Protein efficiently and specifically reactivates pp65-experienced CD4⁺ as well as CD8⁺ T cells. Therefore, CMV pp65 - Recombinant Protein is a useful antigen for the detection and isolation of pp65-experienced CD4⁺ and CD8⁺ effector/memory T cells.